Tokunaga et al. have reported that a particular type of bacterial DNA stimulates an immune response (Yamamoto et al., Jpn. J. Cancer Res. 79:866-873, 1988). A major component of the bacterial DNA essential for an immunostimulatory activity is a characteristic short sequence structure containing a CpG dinucleotide motif (hereinafter abbreviated as CpG) which is not methylated. It has been also reported that a synthesized CpG-containing oligonucleotide induces the production of type I IFN (IFN-α and IFN-β) and IFN-γ in macrophages and natural killer (NK) cells and has a cytotoxic activity of the NK cells (JP Hei-4-352724-A). It has been also reported that the CpG-containing oligonucleotide acts upon not only the macrophages but also dendritic cells and B cells and induces a cell proliferative activity and the production of inflammatory cytokines of interleukin-12 (IL-12), tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) (Klinman et al., Proc. Natl. Acad. Sci., 93:2879-2883, 1996). Thus, the CpG-containing oligonucleotide is useful as an adjuvant of a vaccine and for the treatment of allergic diseases because it induces cellular immunity and Th1 responses. On the other hand, it can not be denied that the CpG-containing oligonucleotide might trigger side effects such as sepsis, fever, joint pain, muscular pain and flare because of inducing the production of TNF-α and IL-6.
Tokunaga et al., have found that the oligonucleotide containing the CpG motif and composed of a 6 bases-palindrome motif has strong activity in the cytotoxic activity in the murine NK cells, and have reported that the sequences of 5′-AACGTT-3′ (SEQ ID NO:92), 5′-AGCGCT-3′ (SEQ ID NO:93) and 5′-GACGTC-3′ (SEQ ID NO:61) have the strongest activity (Yamamoto at el., J. Immunol., 148:4072-4076, 1992). Other types of immunostimulatory oligonucleotide sequences have been reported (International Publication No. 1998/018810 Pamphlet, International Publication No. 2003/015711 Pamphlet, International Publication No. 2004/058179 Pamphlet).
Studies for the purpose of augmenting activity of oligonucleotides have been also conducted. Tokunaga et al., have found that NK cell activities and IFN-inducing activities are augmented when a repeating structure of deoxyguanylic acid (poly-G sequence) is inserted outside the 6 bases-palindrome motif containing CpG (JP Hei-4-352724-A). It has been also demonstrated that the sequence outside the 6 bases-sequence containing the CpG motif has no small effect on the activity.
The other CpG-containing sequences known publicly include D-type (or A-type) and K-type (or B-type) of immunostimulatory oligonucleotides (International Publication No. 2000/61151 Pamphlet). The K-type is known to activate the B cells. The D-type, where a poly-G sequence is added outside the CpG-containing palindrome sequence, induces a production of type I IFN in the dendritic cells and activates human NK cells. It has been described that, for the IFN-inducing activity of the D-type, the 3′ terminal side is important, and 4 or more bases are required for a length of the poly-G sequence at the 3′ terminal (International Publication No. 2000/61151 Pamphlet). Meanwhile, it has been also described that the poly-G sequence at the 3′ terminal side is important for activities of inducing the production of inflammatory cytokines of IL-12 and TNF-α, and a poly-G sequence of at least 4 or more bases is required for eliciting these effects (Korea KR 2001-063153). Therefore, the sequences of the publicly known oligonucleotides which enhance immunostimulatory activities, or the structure of a poly-G sequence does not reveal an independency of the induction of IFN and the induction of inflammatory cytokines.
It has been found that the immunostimulatory nucleotide having the palindrome motif of 5′-GACGATCGTC-3′ (SEQ ID NO:76) has stronger activity of inducing IFN-α than the conventional unmodified CpG-containing immunostimulatory nucleotides (the effect of modified ones will be described later), by inserting a poly-G sequence having an appropriate length up to 10 at the 3′ terminus and the 5′ terminus (JP 2005-237328-A). It has been disclosed that to confer a higher activity of inducing IFN-α to the immunostimulatory nucleotides having 5′-GACGATCGTC-3′ (SEQ ID NO:76), it is better to put 8 to 10 G bases at the 3′ terminus or the 5′ terminus, but to inhibit a production of interleukin-10 (IL-10) which is an immunosuppressive cytokine, it is better to put unevenly the base(s) at the 5′ terminus only (JP 2005-237328-A). It has been also reported that the activity of inducing the inflammatory cytokines of TNF-α and IL-12 is moderately correlated to the activity of inducing INF-α. These do not reveal an effect by insertion of the poly-G sequence at the 5′ terminus in a palindrome sequence other than 5′-GACGATCGTC-3′ (SEQ ID NO:76). An optimal base number of the poly-G sequence of the CpG-containing oligonucleotide, by which the activity to induce an inflammatory cytokine is attenuated and the IFN-inducing activity for both IFN-γ and IFN-α is augmented, is not disclosed.
5′-GGTGCCGATCGGCAGGGGG-3′ (SEQ ID NO:1) has been found as an oligonucleotide having higher immunostimulatory activity than the conventional D-type of CpC-oligonucleotide (JP 2004-287102-A). Derivatives obtained by substituting one to several bases in this base sequence have been also disclosed. Although no specific sequence obtained by substituting 3 or more bases has been disclosed, only one sequence, 5′-GGGGGGTGCCGATCGGCAGGG-3′ (SEQ ID NO:5) obtained by substituting 7 bases has been found to have IFN-inducing activity even when the poly-G sequence at the 3′ terminus is composed of three bases (International Publication No. 2006/035939 Pamphlet).
Concerning the other study for the purpose of augmenting the activity, stabilization of the oligonucleotide by chemical modification has been known. Naturally occurring phosphodiester nucleotides are degraded easily by various nucleic acid degradation activities in cells and in cell cultures. Therefore, it has been studied that, the phosphodiester nucleotides are stabilized by substituting an internucleotide phosphodiester bond which is an attack target of the nucleic acids degradation activity, and the resulting activity is augmented. A substitution frequently used is the substitution to phosphorothioate. The study by Klinman et al has shown that the induction of immune response is augmented by modifying a poly-G sequence outside the palindrome motif with phosphorothioate (International Publication No. 2000/61151 Pamphlet).
It has been found in the study using TLR9 knockout mice that a receptor for the bacterial DNA containing non-methylated CpG is TLR9, which is one member in Toll-like receptor (TLR) family (Hemmi, et al., Nature, 408:740-745, 2000). It has been also shown that the optimal CpG sequences to activate human TLR9 and murine TLR9 are different, indicating the presence of species specificity (Bauer, et al., PNAS, 98(16):9237-9242, 2001). In the development for the purpose of a therapy in human, it is essential that the CpG-oligonucleotide has high affinity to human TLR9. Also, it is important that the CpG-oligonucleotide acts upon animals such as mice in preclinical studies using the animals which are in developmental stage.
For the treatment of allergic diseases, not a symptomatic treatment currently used frequently but an immunoregulatory type and an effective radical treatment have been desired. In the patients with allergy, the immune response to an allergen is leans to Th2 and the Th1 immune response is suppressed. Therefore, a therapeutic agent that induces the Th1 immune response and suppresses the Th2 immune response is useful for improving an allergic predisposition. It has been disclosed that the CpG oligonucleotide composed of a certain non-palindrome sequence (JP 2003-286174-A) and the CpG-oligonucleotide containing the 6 base-palindrome 5′-AACGTT-3′ (SEQ ID NO:92) (JP 2002-500159—, Tighe, et al., J. Allergy Clin. Immunol., 106:124-134, 2000) have therapeutic effects in a murine asthma model. However, the aforementioned immunostimulatory oligonucleotides are concerned to incur unfavorable side effects when administered at a pharmacologically effective amount because these not only induce the Th1 response but also elicit the inflammatory cytokines.
An allergic symptom is caused by extracellularly releasing granules (degranulation) containing histamine from mast cells, and this degranulation is caused by binding the allergen to IgE on the mast cell. In recent years, an antigen-specific regulatory T cell (Treg) has been noted in its function to keep the immune balance. For example, the inhibition of the degranulation by Treg via IgE receptor FcεRI may be included (Till, et al., J. Allergy Clin. Immunol., 113:1025-1034). IFN-α and IFN-β are shown to facilitate the induction of the production of IL-10 (Aman, et al., Blood, 87:4731-4736, 1996). Furthermore, IL-10 promotes the induction of the differentiation of IgG4- and IgA-producing cells. Thus, it is conceivable that the induction of IL-10 is also one of mechanisms in allergy therapeutic effects by the immunostimulatory nucleotide. Therefore, it is desirable that the immunostimulatory nucleotide suitable for the allergy treatment augments the IFN-α-inducing activity and keeps the IL-10-inducing activity.
Hepatitis indicates the disease including hepatic inflammation induced by virus, alcohol, drug, toxin and autoimmunity.
Among them, a majority is the hepatitis caused by hepatitis virus, in particular, A, B and C types are frequent, and additionally the presence of D, E, F, G and idiopathic hepatitis virus has been known. The above viruses distribute in many different viral families of RNA types and DNA types.
Hepatitis B virus and hepatitis C virus cause acute and chronic infection. In acute hepatitis, the symptom appears in an early infection or in recurrence in chronic infected patients. Meanwhile, in chronic hepatitis C, the hepatic inflammation continues for 6 months or longer, and the cells are destroyed, and then hepatic functions are reduced. In the infection with HCV, it is also problematic that a risk to progress from the acute hepatitis to the chronic hepatitis is high. From such a circumstance, early interference by the treatment and a highly effective therapeutic method for hepatitis virus infection disease are desired.
In hepatitis type C, the treatment with various interferon (IFN) formulations alone or the combination treatment using the IFN-α formulation and ribavirin is the first choice of therapeutic means. In the combination treatment, the sustained effect can be expected compared with the treatment with a single medicament, but then, is more expensive and is accompanied with more side effects. However, even when these treatments are given, the therapeutic effect is observed in only about 60% of the total treated patients, and when the treatment is discontinued after the effect was observed, a half or more patients suffer a recurrence. From these circumstances, further developments of therapeutic drugs have been desired.
The therapeutic effects of the CpG oligonucleotide on the hepatitis may include augmentation of anti-viral effect by induction of interferon, the induction of cellular immunity against the cells infected with the virus and resistance to appearance of resistant strains. Concerning the use of the CpG-oligonucleotide for treating the hepatitis due to the infection with HBV or HCV, technical information is available in JP 2003-526662 and JP 2006-515277. The former has disclosed a method of treating without administering the CpG oligonucleotide (immune activation sequence: ISS) together with a hepatitis viral antigen. The latter has disclosed a method of treating individuals with chronic hepatitis type C where an antiviral agent such as interferon was ineffective, and the clinical study result of a developmental number CpG10101 for usefulness in the patients was disclosed in European Association for the Study of Liver Diseases in 2006. However, from this clinical study result, the therapeutic effect of CpG10101 alone is extremely insufficient compared with the conventional therapies. The combination therapy using three agents, pegylated IFN-α, ribavirin and the CpG-oligonucleotide is expected to have a slightly higher effectiveness compared with the result of the standard therapy such as pegylated IFN-α with ribavirin, but has been already demonstrated not to give a sufficient therapeutic effect compared with the combination with the other antiviral agents (e.g., a combination therapy with the inhibitor of a viral enzyme such as polymerase and protease).
It can not be denied that the existing CpC-oligonucleotide might elicit sepsis, fever, joint pain, muscular pain and reddening and unexpected side effects due to inducing the production of TNF-α, IL-12 and IL-6 which are the inflammatory cytokines. In fact, it has been shown that the existing CpG-oligonucleotide deteriorates a symptom of hepatitis in the study example using a murine hepatitis model (Abe, et al., Fukushima J. Med. Sci., 51:41-49, 2005). Therefore, it has been desired to develop CpG-oligonucleotides having the improved immunostimulatory activity suitable for the treatment of hepatitis and whose side effect is reduced.
Depending on an activity of the immunostimulatory oligonucleotides, it becomes possible to reduce a dosage and an administration frequency. As a result, it is highly likely that occurrence of a toxic action is reduced and QOL is improved.
In light of the foregoing, it is very useful and extremely highly beneficial in industrial application to find a sequence by which the IFN-inducing activity is enhanced and the inflammatory cytokine-inducing activity is reduced in human compared with the aforementioned existing immunostimulatory sequence.